GNAS knockdown suppresses osteogenic differentiation of mesenchymal stem cells via activation of Hippo signaling pathway

Bone marrow-derived mesenchymal stem cells (BMSCs) are a suitable option for cell-based tissue engineering therapies due to their ability to renew and differentiate into multiple different tissue types, such as bone. Over the last decade, the effect of GNAS on the regulation of osteoblast differentiation has attracted great attention. Herein, this study aimed to explore the role of GNAS in osteogenic differentiation of MSCs. A total of 85 GNAS^f/f male mice were selected for animal experiments and 10 GNAS^f/f male mice for BMSC isolation to conduct cell experiments. The mice and BMSCs were treated with Verteporfin (a Hippo signaling pathway inhibitor) to inhibit the Hippo signaling pathway or recombinant adenovirus-expressing Cre to knockout the GNAS expression. Next, computed tomography scan, Von Kossa staining, and alizarin red staining were performed to detect osteogenic differentiation ability. Moreover, immunohistochemistry and alkaline phosphatase (ALP) staining were used to assess the expression of Oc and Osx in femur tissues and ALP activity. At last, the expression of GNAS, osteogenic markers, and factors related to the Hippo signaling pathway was evaluated. Initially, the results displayed successful knockout of the GNAS gene from mice and BMSCs. Moreover, the data indicated that GNAS knockout inhibits expression of Oc, Osx, ALP, BMP-2, and Runx2, and ALP activity. Additionally, GNAS knockout promotes activation of the Hippo signaling pathway, so as to repress osteogenic differentiation. Collectively, depleted GNAS exerts an inhibitory role in osteogenic differentiation of MSCs by activating Hippo signaling pathway, providing a candidate mediator for osteoporosis.     

Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells

Multipotential adult mesenchymal stem cells (MSCs) are able to differentiate along several known lineages, and lineage commitment is tightly regulated through specific cellular mediators and interactions. Recent observations of a low/high bone-mass phenotype in patients expressing a loss-/gain-of-function mutation in LRP5, a coreceptor of the Wnt family of signaling molecules, suggest the importance of Wnt signaling in bone formation, possibly involving MSCs. To analyze the role of Wnt signaling in mesenchymal osteogenesis, we have profiled the expression of WNTs and their receptors, FRIZZLEDs (FZDs), and several secreted Wnt inhibitors, such as SFRPs, and examined the effect of Wnt 3a, as a representative canonical Wnt member, during MSC osteogenesis in vitro. WNT11, FZD6, SFRP2, and SFRP3 are upregulated during MSC osteogenesis, while WNT9A and FZD7 are downregulated. MSCs also respond to exogenous Wnt 3a, based on increased beta-catenin nuclearization and activation of a Wnt-responsive promoter, and the magnitude of this response depends on the MSC differentiation state. Wnt 3a exposure inhibits MSC osteogenic differentiation, with decreased matrix mineralization and reduced alkaline phosphatase mRNA and activity. Wnt 3a treatment of fully osteogenically differentiated MSCs also suppresses osteoblastic marker gene expression. The Wnt 3a effect is accompanied by increased cell number, resulting from both increased proliferation and decreased apoptosis, particularly during expansion of undifferentiated MSCs. The osteo-suppressive effects of Wnt 3a are fully reversible, i.e., treatment prior to osteogenic induction does not compromise subsequent MSC osteogenesis. The results also showed that sFRP3 treatment attenuates some of the observed Wnt 3a effects on MSCs, and that inhibition of canonical Wnt signaling using a dominant negative TCF1 enhances MSC osteogenesis. Interestingly, expression of Wnt 5a, a non-canonical Wnt member, appeared to promote osteogenesis. Taken together, these findings suggest that canonical Wnt signaling functions in maintaining an undifferentiated, proliferating progenitor MSC population, whereas non-canonical Wnts facilitate osteogenic differentiation. Release from canonical Wnt regulation is a prerequisite for MSC differentiation. Thus, loss-/gain-of-function mutations of LRP5 would perturb Wnt signaling and depress/promote bone formation by affecting the progenitor cell pool. Elucidating Wnt regulation of MSC differentiation is important for their potential application in tissue regeneration.     

Regulation of the Wnt/beta-catenin pathway by redox signaling

Although it is well established that reactive oxygen species (ROS) can function as intracellular messengers, the mechanism of ROS dependent signaling is largely unknown (Rhee et al.,2005). In a recent paper in Nature Cell Biology, Funato et al. (2006) demonstrate that ROS can modulate signaling by the Wnt/beta-catenin pathway. This work provides interesting new insight into cross-talk between redox and Wnt/beta-catenin signaling in normal physiology and cancer.     

Wnt/Lrp/beta-catenin signaling suppresses adipogenesis by inhibiting mutual activation of PPARgamma and C/EBPalpha

Wnt/beta-catenin signaling has been implicated in repressing adipogenesis. Several lines of evidence show that the possible mechanism is blockade of PPARgamma induction. However, the precise mechanisms remain to be elucidated. In this study, we demonstrated that Wnt3a conditioned medium suppresses C/EBPbeta/delta-induced adipogenesis of 3T3-L1 cells by inhibiting PPARgamma induction. In addition, the mutual activation of PPARgamma and C/EBPalpha was also repressed in the presence of Wnt3a. To further investigate the role of the canonical Wnt pathway in adipogenesis, we used mouse embryonic fibroblasts (MEFs) isolated from Lrp6-deficient embryos. Contrary to wild-type MEFs, Lrp6-deficient MEFs showed spontaneous adipogenesis and escaped the suppressive effect of exogenous Wnt3a. These findings suggest a critical role of Wnt/Lrp6/beta-catenin signaling in adipogenesis and cell fate decision of mesenchymal stem cells.     

Cross-talk between Wnt signaling pathways in human mesenchymal stem cells leads to functional antagonism during osteogenic differentiation

Wnt signaling is involved in developmental processes and in adult stem cell homeostasis. This study analyzes the role(s) of key Wnt signaling mediators in the maintenance and osteogenesis of mesenchymal stem cells (MSCs). We focus specifically on the involvement of low-density lipoprotein-related protein 5 (LRP5), T-cell factor 1 (TCF1), and Frizzled (Fz) receptors, in the presence or absence of exogenous, prototypical canonical (Wnt3a), and non-canonical (Wnt5a) Wnts. In undifferentiated MSCs, LRP5 and TCF1 mediate canonical Wnt signal transduction, leading to increased proliferation, enhanced synergistically by Wnt3a. However, LRP5 overexpression inhibits osteogenic differentiation, further suppressed by Wnt3a. Wnt5a does not affect cell proliferation but enhances osteogenesis of MSCs. Interestingly, Wnt5a inhibits Wnt3a effects on MSCs, while Wnt3a suppresses Wnt5a-mediated enhancement of osteogenesis. Flow cytometry revealed that LRP5 expression elicits differential changes in Fz receptor profiles in undifferentiated versus osteogenic MSCs. Taken together, these results suggest that Wnt signaling crosstalk and functional antagonism with the LRP5 co-receptor are key signaling regulators of MSC maintenance and differentiation.     

CLCA1 suppresses colorectal cancer aggressiveness via inhibition of the Wnt/beta-catenin signaling pathway

Background

Chloride channel accessory 1 (CLCA1) belongs to the calcium-sensitive chloride conductance protein family, which is mainly expressed in the colon, small intestine and appendix. This study was conducted to investigate the functions and mechanisms of CLCA1 in colorectal cancer (CRC).

Methods

The CLCA1 protein expression level in CRC patients was evaluated by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and western blotting analysis. Using CRISPR/Cas9 technology, CLCA1-upregulated (CLCA1-ACT) and CLCA1-knockout cells (CLCA1-KO), as well as their respective negative controls (CLCA1-ACT-NC and CLCA1-KO-NC), were constructed from the SW620 cell line. Cell growth and metastatic ability were assessed both in vitro and in vivo. The association of CLCA1 with epithelial-mesenchymal transition (EMT) and other signaling pathways was determined by western blotting assays.

Results

The expression level of CLCA1 in CRC tissues was significantly decreased compared with that in adjacent normal tissue (P< 0.05). Meanwhile, the serum concentration of CLCA1 in CRC patients was also significantly lower when compared with that of healthy controls (1.48?±?1.06 ng/mL vs 1.06?±?0.73 ng/mL, P?=?0.0018). In addition, CLCA1 serum concentration and mRNA expression level in CRC tissues were inversely correlated with CRC metastasis and tumor stage. Upregulated CLCA1 suppressed CRC growth and metastasis in vitro and in vivo, whereas inhibition of CLCA1 led to the opposite results. Increased expression levels of CLCA1 could repress Wnt signaling and the EMT process in CRC cells.

Conclusions

Our findings suggest that increased expression levels of CLCA1 can suppress CRC aggressiveness. CLCA1 functions as a tumor suppressor possibly via inhibition of the Wnt/beta-catenin signaling pathway and the EMT process.

     

Vibration loading promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells via p38 MAPK signaling pathway

Low magnitude high frequency vibration (LMHFV) exhibits effectively anabolic effects on the bone tissue, and can promote osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. The role of p38 MAPK signaling in LMHFV-induced osteogenesis remains unclear. In this current study, LMHFV loading was applied to BMSCs in vitro, and cell proliferation, alkaline phosphatase (ALP), matrix mineralization, as well as osteogenic genes expression were assayed. The mechanism of mechanical signal transduction was analysed using PCR array, qRT-PCR and Western blot. LMHFV increased cell proliferation in the growth medium, while inhibited proliferation in the osteogenic medium. ALP activity, matrix mineralization and osteogenic genes expression of Runx2, Col-I, ALP, OPN and OC were increased by LMHFV. p38 and MKK6 genes expression, and p38 phosphorylation were promoted in LMHFV-induced osteogenesis. Inhibition of p38 MAPK with SB203580 and targeted p38 siRNA blunted the increased ALP activity and osteogenic genes expression by LMHFV. These findings suggest that LMHFV promotes osteogenic differentiation of BMSCs, and p38 MAPK signaling shows an important function in LMHFV-induced osteogenesis.     

Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis

Chondrocytes and osteoblasts are two primary cell types in the skeletal system that are differentiated from common mesenchymal progenitors. It is believed that osteoblast differentiation is controlled by distinct mechanisms in intramembranous and endochondral ossification. We have found that ectopic canonical Wnt signaling leads to enhanced ossification and suppression of chondrocyte formation. Conversely, genetic inactivation of beta-catenin, an essential component transducing the canonical Wnt signaling, causes ectopic formation of chondrocytes at the expense of osteoblast differentiation during both intramembranous and endochondral ossification. Moreover, inactivation of beta-catenin in mesenchymal progenitor cells in vitro causes chondrocyte differentiation under conditions allowing only osteoblasts to form. Our results demonstrate that beta-catenin is essential in determining whether mesenchymal progenitors will become osteoblasts or chondrocytes regardless of regional locations or ossification mechanisms. Controlling Wnt/beta-catenin signaling is a common molecular mechanism underlying chondrocyte and osteoblast differentiation and specification of intramembranous and endochondral ossification.     

ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice

Osteogenic differentiation refers to the process of bone formation and remodeling, which is controlled by complex molecular mechanisms. Activin A receptor type I (ACVR1) is reported to be associated with osteogenic differentiation. However, the underlying molecular mechanism remains elusive. Therefore, this study evaluates the function of ACVR1 in osteogenic differentiation through the Wnt signaling pathway. The expression of osteocalcin (Oc) and osterix together with osteogenic differentiation and mineralization was examined in ACVR1-knockout (KO) mouse. Furthermore, the Wnt signaling pathway was inhibited in bone marrow stromal cells (BMSCs) of mice to explore the role of the Wnt signaling pathway in osteogenic differentiation by means of alkaline phosphatase (ALP) activity detection and evaluation of mineralized nodules and calcium content. Subsequently, the effect of ACVR1 on the Wnt signaling pathway was assessed by determining the expression of ACVR1, β-catenin, glycogen synthase kinase 3 β (GSK3β), dickkopf-related protein 1 (DKK1), and frizzled class receptor 1 (FZD1). Both their effects on osteogenic differentiation were further evaluated by determination of Oc, osterix, and Runx2 expression. AVCR1 KO mice exhibited increased Oc and osterix expression and promoted bone resorption and formation. ACVR1-knockout was observed to activate the Wnt signaling pathway with an increase of β-catenin and reductions in GSK3β, DKK1, and FZD1. With the inhibited Wnt signaling pathway expression of Oc, osterix, and Runx2 was decreased, and ALP activity, mineralized nodule, and calcium content in cellular matrix were decreased as well, indicating that inactivation of the Wnt signaling pathway reduced the differentiation of BMSCs into osteoclasts. These findings indicate that ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice.     

Regulation of beta-catenin signaling in the Wnt pathway

beta-Catenin not only regulates cell to cell adhesion as a protein interacting with cadherin, but also functions as a component of the Wnt signaling pathway. The Wnt signaling pathway is conserved in various organisms from worms to mammals, and plays important roles in development, cellular proliferation, and differentiation. Wnt stabilizes cytoplasmic beta-catenin and then beta-catenin is translocated into the nucleus where it stimulates the expression of genes including c-myc, c-jun, fra-1, and cyclin D1. The amounts and functions of beta-catenin are regulated in both the cytoplasm and nucleus. Its molecular mechanisms are becoming increasingly well understood.     

The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells

Neural precursor cells (NPCs) have the ability to self-renew and to give rise to neuronal and glial lineages. The fate decision of NPCs between proliferation and differentiation determines the number of differentiated cells and the size of each region of the brain. However, the signals that regulate the timing of neuronal differentiation remain unclear. Here, we show that Wnt signaling inhibits the self-renewal capacity of mouse cortical NPCs, and instructively promotes their neuronal differentiation. Overexpression of Wnt7a or of a stabilized form of beta-catenin in mouse cortical NPC cultures induced neuronal differentiation even in the presence of Fgf2, a self-renewal-promoting factor in this system. Moreover, blockade of Wnt signaling led to inhibition of neuronal differentiation of cortical NPCs in vitro and in the developing mouse neocortex. Furthermore, the beta-catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized beta-catenin did not induce neuronal differentiation of cortical NPCs at earlier developmental stages, consistent with previous reports indicating self-renewal-promoting functions of Wnts in early NPCs. These findings may reveal broader and stage-specific physiological roles of Wnt signaling during neural development.     

NT-3 promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells by regulating the Akt pathway

Objectives:To investigate the effect of neurotrophin-3 (NT-3) on osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Methods:Osteogenic differentiation was detected by alkaline phosphatase (ALP) staining and alizarin red staining (ARS). Adipogenic differentiation was detected by oil red O (ORO) staining. The expression of bone-related genes (Runx2, Osterix, OCN, ALP) and lipogenic genes (FABP4, PPAR, CEBP, LPL) was detected by real-time quantitative polymerase chain reaction (real-time qPCR). The expression of p-Akt and Akt protein was detected by Western blot assay.Results:ALP staining and ARS staining showed that the overexpression of NT-3 could promote the differentiation into osteoblasts, while knockdown of NT-3 could inhibit that. Real-time qPCR showed that the overexpression of NT-3 could increase the expression of osteoblast genes, while knockdown of NT-3 could inhibit that. ORO staining showed that the overexpression of NT-3 could inhibit the differentiation into adipogenesis, while knockdown of NT-3 can promote that. Real-time qPCR showed that the overexpression of NT-3 could reduce the expression of lipogenic genes. while knockdown NT-3 could increase that. In addition, the overexpression of NT-3 increased p-Akt/Akt levels significantly, while knockdown NT-3 reduced that significantly.Conclusion:NT-3 could promote the differentiation of mouse BMSCs into osteoblasts and inhibit their differentiation into adipogenesis.     

Uniaxial mechanical tension promoted osteogenic differentiation of rat tendon-derived stem cells (rTDSCs) via the Wnt5a-RhoA pathway

Chronic tendinopathy is a tendon disorder that is common in athletes and individuals whose tendons are subjected to repetitive strain injuries. The presence of ossification worsened the clinical manifestation of the disorder. The change of tendon loading due to mechanical overload, compression, or disuse have been implicated as the possible etiologies, but the pathological mechanisms of tendinopathy remain unclear. In this study, we demonstrated that ossification in tendon tissue might be due to the osteogenesis of tendon‐derived stem cells (TDSCs) induced by uniaxial mechanical tension (UMT) which mimics the mechanical loading in tendon. Rat TDSCs (rTDSCs) could be induced to differentiate into osteogenic lineage after treatment with 2% elongation UMT for 3 days as shown by the increased expression Runx2 mRNA and protein, Alpl mRNA, collagen type 1 alpha 1 (Col1a1) mRNA, ALP activity, and ALP cytochemical staining. RhoA, an osteogenesis regulator, was activated in rTDSCs upon UMT stimulation. Blockage of RhoA activity in rTDSCs by C3 toxin or ROCK activity, a downstream target of RhoA, by Y‐27632 inhibited UMT‐induced osteogenesis in rTDSCs. UMT up‐regulated the mRNA expression of Wnt5a but not the other non‐canonical Wnts. The inhibition of Wnt5a expression by siRNA abolished UMT‐induced Runx2 mRNA expression and RhoA activation in rTDSCs and the inhibition of Runx2 expression could be rescued by addition of LPA, a RhoA activator. In conclusion, our results showed that UMT induced osteogenic differentiation of rTDSCs via the Wnt5a‐RhoA pathway, which might contribute to ectopic ossification in tendon tissue due to mechanical loading. J. Cell. Biochem. 113: 3133–3142, 2012. © 2012 Wiley Periodicals, Inc.